Comprehensive evaluation of factors influencing selenium fertilization biofortification.

BACKGROUND: Dietary selenium (Se) deficiency, stemming from low Se concentrations in agricultural products, threatens human health. While Se-containing fertilizers can enhance the Se content in crops, the key factors governing Se biofortification with Se fertilization remain unclear. RESULTS: This study constructed a global meta-analysis dataset based on field experiments comprising 364 entries on Se content in agricultural products and 271 entries on their yield. Random forest models and mixed effects meta-analyses revealed that plant types (i.e., cereals, vegetables, legumes, and forages) primarily influenced Se biofortification, with Se fertilization rates being the next significant factor. The random forest model, which included variables like plant types, Se fertilization rates, methods and types of Se application, initial soil conditions (including Se content, organic carbon content, and pH), soil types, mean annual precipitation, and temperature, explained 82.14% of the variation in Se content and 48.42% of the yield variation in agricultural products. For the same agricultural products, the increase in Se content decreased with higher rates of Se fertilization. The increase in Se content in their edible parts will be negligible for cereals, forages, legumes, and vegetable crops, when Se fertilization rates were 164, 103, 144, and 147 g Se ha-1 , respectively. Conversely, while low Se fertilization rates enhanced yields, high rates led to a yield reduction, particularly in cereals. CONCLUSION: Our findings highlight the need for balanced and precise Se fertilization strategies to optimize Se biofortification benefits and minimize the risk of yield reduction. © 2024 Society of Chemical Industry.

Selenium loss during boiling processes and its bioaccessibility in different crops: Estimated daily intake.

Food crops provide a good selenium (Se) source for Se-deficient populations. This study assessed how boiling affects Se concentration, speciation, and bioaccessibility in common food crops to determine human Se intake. Boiling rice resulted in an 11.9% decrease in minimum Se content, while sorghum experienced a maximum (34.9%) reduction. Boiled vegetables showed a 21% - 40% Se loss. Cereals showed notable decreases in selenomethionine (SeMet) and selenocysteine (SeCys2), while most vegetables exhibited a significant reduction in Se-methylselenocysteine (SeMeCys). Boiling significantly reduced the Se bioaccessibility in all food crops, except cabbage and potato. Cereal crops were more efficacious in meeting the recommended daily intake (RDI) of Se compared to vegetables. Rice exceeds other crops and provides up to 39.2% of the WHO/FAO-recommended target minimum daily intake of 60 µg/day. This study provides insight into a substantial dissonance between the estimated daily intake (EDI) of Se and the bioaccessible Se in both raw and boiled crops. Consequently, revising EDI standards is imperative.

Characterization of Selenium Speciation in Se-Enriched Crops: Crop Selection Approach.

Accurately quantifying selenium (Se) speciation and transformation in Se-enriched crops is highly significant for human health. The investigation of Se species in Se-enriched crops involves assessing the enrichment of both organic and inorganic Se species, considering their plant families and edible parts. The staple crops of rice, corn, and wheat showed no or less inorganic Se with the increase of total Se; however, potatoes expressed a proportion of selenate [Se(VI)]. In addition, the organic Se proportions in Se-enriched crops of Cruciferous, Brassicaceae, and Umbelliferae plant families were relatively lower than the proportion of inorganic Se. Concurrently, the edible parts of the Se-enriched gramineous or cereal crops enriched with organic Se and crops with fruit, stem, leaf, and root as edible parts contain the maximum percentage of organic Se with a certain proportion of inorganic Se. This study contributes to a sparse body of literature by meticulously discerning appropriate Se-enriched crop selection through a comprehensive evaluation of Se speciation and its organic and inorganic accumulation potential.

In silico design of a broad-spectrum multiepitope vaccine against influenza virus.

Influenza remains a global health concern due to its potential to cause pandemics as a result of rapidly mutating influenza virus strains. Existing vaccines often struggle to keep up with these rapidly mutating flu viruses. Therefore, the development of a broad-spectrum peptide vaccine that can stimulate an optimal antibody response has emerged as an innovative approach to addressing the influenza threat. In this study, an immunoinformatic approach was employed to rapidly predict immunodominant epitopes from different antigens, aiming to develop an effective multiepitope influenza vaccine (MEV). The immunodominant B-cell linear epitopes of seasonal influenza strains hemagglutinin (HA) and neuraminidase (NA) were predicted using an antibody-peptide microarray, involving a human cohort including vaccinees and infected patients. On the other hand, bioinformatics tools were used to predict immunodominant cytotoxic T-cell (CTL) and helper T-cell (HTL) epitopes. Subsequently, these epitopes were evaluated by various immunoinformatic tools. Epitopes with high antigenicity, high immunogenicity, non-allergenicity, non-toxicity, as well as exemplary conservation were then connected in series with appropriate linkers and adjuvants to construct a broad-spectrum MEV. Moreover, the structural analysis revealed that the MEV candidates exhibited good stability, and the docking results demonstrated their strong affinity to Toll-like receptors 4 (TLR4). In addition, molecular dynamics simulation confirmed the stable interaction between TLR4 and MEVs. Three injections with MEVs showed a high level of B-cell and T-cell immune responses according to the immunological simulations in silico. Furthermore, in-silico cloning was performed, and the results indicated that the MEVs could be produced in considerable quantities in Escherichia coli (E. coli). Based on these findings, it is reasonable to create a broad-spectrum MEV against different subtypes of influenza A and B viruses in silico.

A preliminary predictive model for selenium nutritional status in residents based on three selenium biomarkers.

BACKGROUND: Selenium (Se) is an essential nutrient and an important component of many selenoproteins that possess fundamental importance to human health. Selenium deficiency and excess will cause corresponding diseases in the human body. The nutritional health of Se in the human body mainly depends on the daily dietary Se intake of the human body, which in turn depends to a certain extent on the content of Se transmitted along the food chain. This study aims to research the transport of Se through the soil-crop-human chain in regions with different Se levels, and to establish the model between the residents' dietary Se intake and the three Se biomarkers (hair, nails, and plasma), to predict the nutritional health status of Se in residents through Se biomarkers. METHOD: Carry out field and cross-sectional surveys of populations in Loujiaba Village and Longshui Village. Samples were collected from soil, crops, drinking water, residents' hair, nails, plasma, and diet. The concentration of available Se fractions was extracted from soil samples using 0.1 mol/L K2HPO4. The concentration of total Se for all samples was determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), and the relative standard deviation was less than 5%. In this study, hair, nails, and blood samples were collected from volunteers according to the Declaration of Helsinki and the Ethics Committee of Soochow University. The dietary nutritional structure and dietary Se intake of the population were randomly selected by 12 volunteers using the duplicate portion method. Data were described using mean ± standard deviation. We performed saliency analysis and correlation analysis (with Pearson correlation coefficient), and fitted a regression to evaluate the associations between these variables. RESULTS: The soil total Se (5201 ± 609.2 µg/kg) and available Se (307.7 ± 83.5 µg/kg) in Luojiaba Village (LJB) were significantly higher than the soil total Se (229.2 ± 32.5 µg/kg) and available Se (21.9 ± 4.0 µg/kg) in Longshui Village (LS). The residents' dietary Se intake of LJB (150.3 ± 2.2 µg/d) was within the World Health Organization (WHO) recommended intake range, while LS (16.0 ± 0.4 µg/d) was close to the range of Keshan disease occurrence, and there was a risk of insufficient Se intake. The correlation analysis found significant positive correlations between residents' dietary Se intake and the three Se biomarkers. According to the preliminary model established in this study, if the daily dietary Se intake of residents reaches the WHO recommended value of 55-400 µg, the hair, nails, and plasma of Se concentration will be 522.1-2850.5 µg/kg, 1069.0-6147.4 µg/kg, and 128.3-661.36 µg/L, respectively. CONCLUSION: Selenium is transmitted through the soil-crop-human chain, and the Se concentration that enters the human body through the food chain in high-Se areas is significantly higher than that in low-Se areas. The nutritional health status of Se in the human body depends on the daily dietary intake of the human body, and there is a significant correlation between the daily dietary Se intake of the human body and the three biomarkers of Se levels in the human body, so the three biomarkers can be used to evaluate the Se nutritional health of the human.

Selenium Effect Threshold for Soil Nematodes Under Rice Biofortification.

Crop biofortification with inorganic selenium (Se) fertilizer is a feasible strategy to improve the health of residents in Se-deficient areas. For eco-friendly crop Se biofortification, a comprehensive understanding of the effects of Se on crop and soil nematodes is vital. In this study, a rice pot experiment was carried out to test how selenite supply (untreated control (0), 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 200 mg Se kg-1) in soil affected rice growth, rice Se accumulation, and soil nematode abundance and composition. The results showed that selenite supply (5-200 mg kg-1) generally increased the number of rice tillers, rice yield, and Se concentrations in rice grains. In soil under 10 mg kg-1 Se treatment, the genus composition of nematodes changed significantly compared with that in the control soil. With increased Se level (> 10 mg kg-1), soil nematode abundance decreased significantly. Correlation analysis also demonstrated the positive relationships between soil Se concentrations (total Se and bioavailable Se) with rice plant parameters (number of rice tillers, rice yield, and grain Se concentration) and negative relationships between soil Se concentrations (total Se and bioavailable Se) with soil nematode indexes (nematode abundance and relative abundance of Tobrilus). This study provides insight into balancing Se biofortification of rice and soil nematode community protection and suggests the effective concentrations for total Se (1.45 mg kg-1) and bioavailable Se (0.21 mg kg-1) to soil nematode abundances at 20% level (EC20) as soil Se thresholds. At Se concentrations below these thresholds, rice plant growth and Se accumulation in the grain will still be promoted, but the disturbance of the soil nematodes would be negligible.

Influence of Diet on the Effect of the Probiotic Lactobacillus paracasei in Rats Suffering From Allergic Asthma.

Mounting evidence suggests that probiotics can be used to treat allergic asthma by modulating the gut microbiota, and that the effects of probiotics may be influenced by environmental factors such as diet. We conducted a rat model with allergic asthma (AA) modulated by Lactobacillus paracasei, feeding up with high-fat or high-fiber diets based on collecting data from 85 questionnaires. The systemic proinflammatory cytokines were detected by ELISA and the overall structure of fecal microbiota was analyzed via 16S rRNA gene sequencing. The results showed consumption of a high-fiber diet alleviated the allergic symptoms and airway inflammation, and led to improving the imbalance of T-helper type 1 (Th1)/Th2 cells with increased expression of interferon-γ and decreased expression of interleukin-4. Whereas, the high-fat diet had deteriorating implications and skewed the inflammatory perturbation. Furthermore, abundances of phylum Bacteroidetes, families Muribaculaceae, Tannerellaceae, Prevotellaceae, Enterococcaceae, genera Allobaculum, Parabacteroides, and Enterococcus were enriched in L. paracasei-modulating rats fed with high-fiber diet. Firmicutes and Proteobacteria, families Lachnospiraceae, Ruminococcaceae and Desulfovibrionaceae, genera Blautia, unidentified_Ruminococcaceae, unidentified_Clostridiales and Oscillibacter were in relatively high abundance in the rats administered high-fat diet. Association between changed microbiota and inflammatory cytokines was also conferred. These data indicated that the efficacy of L. paracasei in allergic asthma was influenced by different dietary patterns. Hence, diet is important for probiotic therapy when managing allergic asthma.

Soil Selenium Concentration and Residents Daily Dietary Intake in a Selenosis Area: A Preliminary Study in Yutangba Village, Enshi City, China.

In 1963, selenosis occurred in Yutangba Village, Enshi City, China. Subsequently, local residents migrated to a new area of Yutangba to avoid high selenium (Se) exposure. In this study, 19 soil samples, 43 food samples, 60 hair samples and 58 plasma samples from local residents were randomly collected in New Yutangba Village. The mean total Se concentrations in cultivated soil samples were 1753.6 ± 742.8 µg/kg (n = 14). The estimated daily Se intake in New Yutangba Village decreased to 63.2 ± 39.8 µg/day, slightly higher than the recommended dietary Se intake for adults in China (60 µg/day). The mean Se concentrations in hair and plasma samples were 549.7 ± 165.2 µg/kg (n = 60) and 98.4 ± 32.1 µg/L (n = 58), respectively. The result indicated that appropriate activities, such as relocation, consuming a mixture of local foods and market foods containing low Se concentration, could effectively reduce the risk of high Se exposure.

XY-Meta: A High-Efficiency Search Engine for Large-Scale Metabolome Annotation with Accurate FDR Estimation.

FDR control has been a huge challenge for large-scale metabolome annotation. Although recent research indicated that the target-decoy strategy could be implemented to estimate FDR, it is hard to perform FDR control due to the difficulty of getting a reliable decoy database because of the complex fragmentation mechanism of metabolites and ubiquitous isomers. To tackle this problem, we developed a decoy generation method, which generates forged spectra from the reference target database by preserving the original reference signals to simulate the presence of isomers of metabolites. Benchmarks on GNPS data sets in Passatutto showed that the decoy database generated by our method is closer to the actual FDR than other methods, especially in the low FDR range (0-0.05). Large-scale metabolite annotation on 35 data sets showed that strict FDR reduced the number of annotated metabolites but increased the spectral efficiency, indicating the necessity of quality control. We recommended that the FDR threshold should be set to 0.01 in large-scale metabolite annotation. We implemented decoy generation, database search, and FDR control into a search engine called XY-Meta. It facilitates large-scale metabolome annotation applications.

Molecular characterisation of phenylketonuria in a Chinese mainland population using next-generation sequencing.

Phenylketonuria (PKU) is an inherited autosomal recessive disorder of phenylalanine metabolism, mainly caused by a deficiency of phenylalanine hydroxylase (PAH). The incidence of various PAH mutations differs among race and ethnicity. Here we report a spectrum of PAH mutations complied from 796 PKU patients from mainland China. The all 13 exons and adjacent intronic regions of the PAH gene were determined by next-generation sequencing. We identified 194 different mutations, of which 41 are not reported before. Several mutations reoccurred with high frequency including p.R243Q, p.EX6-96A > G, p.V399V, p.R241C, p.R111*, p.Y356*, p.R413P, and IVS4-1G > A. 76.33% of mutations were localized in exons 3, 6, 7, 11, 12. We further compared the frequency of each mutation between populations in northern and southern China, and found significant differences in 19 mutations. Furthermore, we identified 101 mutations that are not reported before in Chinese population, our study thus broadens the mutational spectrum of Chinese PKU patients. Additionally, 41 novel mutations will expand and improve PAH mutation database. Finally, our study offers proof that NGS is effective, reduces screening times and costs, and facilitates the provision of appropriate genetic counseling for PKU patients.

Correlation between genotype and the tetrahydrobiopterin-responsive phenotype in Chinese patients with phenylketonuria.

BACKGROUND: A growing body of research has suggested that tetrahydrobiopterin (BH4) responsive phenotype can be predicted by the phenylalanine hydroxylase (PAH) genotype in patients with phenylketonuria (PKU), but data concerning the association between genotype and BH4 responsiveness are scarce in China. METHODS: A total of 165 PKU patients from China who had undergone a 24-h loading test with BH4 administration were recruited. Genotyping was performed by the next-generation sequencing (NGS) technique. Using the predicted residual PAH activity, we analyzed the association between genotype and BH4-responsiveness. RESULTS: Among the 165 patients, 40 patients (24.24%) responded to BH4. A total of 74 distinct mutations were observed, including 13 novel mutations. The mutation p.R241C was most frequently associated with response. Two known mutations (p.A322T and p.Q419R) and two novel mutations (p.L98V and IVS3-2A>T) were first reported as responsive to BH4. Residual PAH activity of at least 12.5% was needed for responsive genotypes. CONCLUSION: Genotype-based predictions of BH4-responsiveness are only for selecting potential responders. Accordingly, it is necessary to test potential responders with a long-term BH4 challenge.

Mutational spectrum of phenylketonuria in Jiangsu province.

UNLABELLED: Phenylketonuria (PKU) is caused by variants in the phenylalanine hydroxylase (PAH) gene. We systematically investigated all 13 exons of the PAH gene and their flanking introns in 31 unrelated patients and their parents using next-generation sequencing (NGS). A total of 33 different variants were identified in 58 of 62 mutant PAH alleles. The prevalent variants with a relative frequency of 5 % or more were c.721C > T, c.1068C > A, c.611A > G, c.1197A > T, c.728G > A, c.331C > T, and c.442-1G > A. One novel variant was identified in this study-c.699C > G. We studied genotype-phenotype correlations using the Guldberg arbitrary value (AV) system, which revealed a consistency rate of 38 % (8/21) among the 21 predicted phenotypes. The genotype-based prediction of BH4 responsiveness was also evaluated, and 14 patients (45.2 %) were predicted to be BH4 responsive. CONCLUSION: This study presents the spectrum of PAH variants in Jiangsu province. The information obtained from the genotype-based prediction of BH4 responsiveness might be used for the rational selection of candidates for BH4 testing. WHAT IS KNOWN: • Phenylketonuria (PKU) is caused by variants in the phenylalanine hydroxylase (PAH) gene. • The spectrum of PAH variants in different Chinese populations has been reported. What is new: • This is the first report on the spectrum of PAH variants in Jiangsu province. • This study identified one novel PAH variant-c.699C>G-and and tries to show a genotype-phenotype relationship also regarding BH4-responsiveness.

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease.

PURPOSE: This article demonstrates a prominent noninvasive prenatal approach to assist the clinical diagnosis of a single-gene disorder disease, maple syrup urine disease, using targeted sequencing knowledge from the affected family. METHODS: The method reported here combines novel mutant discovery in known genes by targeted massively parallel sequencing with noninvasive prenatal testing. RESULTS: By applying this new strategy, we successfully revealed novel mutations in the gene BCKDHA (Ex2_4dup and c.392A>G) in this Chinese family and developed a prenatal haplotype-assisted approach to noninvasively detect the genotype of the fetus (transmitted from both parents). CONCLUSION: This is the first report of integration of targeted sequencing and noninvasive prenatal testing into clinical practice. Our study has demonstrated that this massively parallel sequencing-based strategy can potentially be used for single-gene disorder diagnosis in the future.

Significance of low serum vitamin D for infection risk, disease severity and mortality in critically ill patients.

BACKGROUND: Hospitalized patients often have higher rate of vitamin D deficiency than healthy people. Vitamin D levels below normal are associated with hospital stay, increased incidence of adverse prognosis and increased mortality of a number of diseases. Whether there is a relationship between vitamin D levels and infection or sepsis in the critically ill is still unclear. This study will explore the relationship between vitamin D levels and risk of infection, assessment for disease severity, and predictor of mortality. METHODS: To evaluate the value of vitamin D in intensive care unit (ICU) cases to sepsis, severity and prognosis assessment, high performance liquid chromatography and tandem mass spectrometry were used to measure the concentrations of vitamin D in sera of critically ill patients. The serum samples were drawn within the first 24 hours of ICU admission. RESULTS: The study included 206 people, 50 healthy controls, 51 ICU control patients and 105 ICU diagnosed with sepsis. Critically ill ICU patients (ICU sepsis and ICU control group) had lower vitamin D concentration than normal people, but septic patients showed no significant reduction of vitamin D concentration when compared with critically ill patients with no positive etiological evidence. For assessment of disease severity, there were very low negative correlations between APACHE II, SAPS II and SOFA scores and vitamin D level. Additionally, patients of different 25-(OH)D levels showed no difference whether in terms of 28-day survival (X(2) = 1.78, P = 0.776) or 90-day survival (X(2) = 4.12, P = 0.389). Multivariate Logistic regression demonstrated that APECHE II and SAPS II scores were independent risk factors to deaths caused by sepsis. CONCLUSION: Clinically, serum concentration of vitamin D is not an indicator for diagnosis and assessment in critically ill patients (ClinicalTrial.gov identifier NCT01636232).

An improvement of shotgun proteomics analysis by adding next-generation sequencing transcriptome data in orange.

BACKGROUND: Shotgun proteomics data analysis usually relies on database search. Because commonly employed protein sequence databases of most species do not contain sufficient protein information, the application of shotgun proteomics to the research of protein sequence profile remains a big challenge, especially to the species whose genome has not been sequenced yet. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we present a workflow with integrated database to partly address this problem. First, we downloaded the homologous species database. Next, we identified the transcriptome of the sample, created a protein sequence database based on the transcriptome data, and integtrated it with homologous species database. Lastly, we developed a workflow for identifying peptides simultaneously from shotgun proteomics data. CONCLUSIONS/SIGNIFICANCE: We used datasets from orange leaves samples to demonstrate our workflow. The results showed that the integrated database had great advantage on orange shotgun proteomics data analysis compared to the homologous species database, an 18.5% increase in number of proteins identification.

Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli.

Large scale production of recombinant human flotillin-2 (reggie-1) is desirable for structural and biochemical studies. However, as the major lipid rafts specific hydrophobic protein, flotillin-2 was difficult to be expressed as soluble and functional form in prokaryotic system. In this study, we first cloned and expressed human flotillin-2 in Escherichia coli with five different fusion tags: poly-histidine, glutathione S-transferase (GST), thioredoxin (TRX), N-Utilization substance (NusA) and maltose binding protein (MBP). We screened the expression level and solubility of the five flotillin-2 fusion proteins, the best MBP tagged flotillin-2 was then large scale produced. The optimized purification procedure included two steps of chromatography: Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 36.0 mg soluble and functional recombinant flotillin-2 from 1 L of culture medium with purity above 97%. The activity of recombinant flotillin-2 was verified by pull-down assay with flotillin-1, showing that the purified recombinant flotillin-2 can specifically interact with flotillin-1. The circular dichroism (CD) spectroscopy showed that recombinant flotillin-2 had a very stable secondary structure dominated by α-helix, ß-turn and random structure.

Expression and purification of recombinant arginine decarboxylase (speA) from Escherichia coli.

The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase's structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His(10)-ADC (77.3 kDa) was first purified by Ni-NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His(10) tag removed recombinant ADC (74.1 kDa)'s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6-9) and temperature (20-80 degrees C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.

Crystallization and preliminary X-ray analysis of fluorescent protein mBanana.

mBanana is a novel monomeric red fluorescent protein mutant. It was cloned and expressed in Escherichia coli with 10 histidine residues at its N-terminal. After cleavage of the His tag by TEV protease, the mBanana was further purified and crystallized by the hanging-drop vapor-diffusion technique. The crystals can diffract to 2.0A resolution and one set of completed data was collected. It showed that the orthorhombic mBanana crystal was in space group P21 with unit cell parameters (48.629, 42.667, 61.714, 90, 111.676, 90) and contained one molecule in one asymmetric unit.

Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.

We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or pT7His (His(10) -mDsRed) prokaryotic expression vectors. Then the fluorescent proteins were expressed in Rosetta (DE3) pLysS by IPTG induction or autoinduction. We purified the fluorescent proteins by affinity chromatography (Amylose and metal ion-chelating column), anion-exchange chromatography (High Q column), size exclusive chromatography (Sephacryl S-200 column), and hydrophobic interaction chromatography (Methyl HIC column) to exhibit the protein-purification techniques. After purification, the fusion protein MBP-EGFP was cleaved by TEV protease. The recombinant mDsRed protein was crystallized by hanging drop vapor diffusion technique to show students the basic operation of crystallization. The whole procedure can be monitored real time by naked eyes when using fluorescent proteins. The demonstration of expression, purification, crystallization, and protease cleavage became much more vivid and interesting, which greatly deepened the students' understanding of modern protein-science techniques.